Stroke is the second leading cause of death worldwide and the primary cause of serious, long-term disability and nursing home admissions. The absence of clinically useful diagnostics has severely impeded effective management and treatment of acute ischemic stroke. Currently available diagnostics are either expensive and/or unreliable and there are no simple, rapid, non-invasive tests to diagnose and assess the magnitude of acute stroke, as well as to predict outcome. Immunoassay technology, such as the enzyme-linked immunosorbent assay (ELISA), can be employed to develop this much needed stroke diagnostic test. Such a test would also help predict the efficacy of potential therapies to treat injury, as well as provide information on precise biochemical and molecular mechanisms contributing to injury and recovery of function. We have developed a specific and sensitive biomarker for acute ischemic stroke. all-Spectrin is a major substrate for calpain and caspase-3 proteolysis, which each produce a unique 150 kDa spectrin breakdown product (SBDP150) and a unique 120 kDa proteolytic fragment (SBDP120), respectively. Work from our laboratories demonstrates that in addition to calpain activation, caspase-3 activation is a robust event following acute ischemic stroke. In this Phase I proposal, we propose to develop a sandwich ELISA that detects the caspase-3 mediated SBDP120. In addition, we are completing development of an ELISA that detects the calpain mediated SBDP150. A major strength of this proposal will be the concurrent and differential detection of SBPD120 measuring apoptosis and SPDP150 measuring oncosis (necrosis) and apoptosis following acute ischemic stroke, allowing important insights into the relative contributions of these two cell death pathways to ischemic injury. Specific Aim 1: Configuration of a sandwich ELISA for the quantitative and specific detection of a caspase-3 mediated spectrin breakdown product of 120 kDa (SBDP120), which is uniquely associated with neuronal apoptosis. Deliverable from Specific Aim 1: Configuration of a neuronal apoptosis-linked diagnostic ELISA kit that quantitatively detects SBDP120. (Months 0-4) Specific Aim 2: Preliminary validation of both the SBDP120 sandwich ELISA and the SBDP150 sandwich ELISA in a rat model of stroke injury. Deliverable from Specific Aim 2: Prototype diagnostic ELISA capable of measuring levels of apoptosis-linked SBDP120 in CSF and serum. In addition, both the SBDP150 and SBDP120 ELISA will be validated in CSF and serum in a rodent model of stroke injury (MCAO injury). (Months 4-6)